JPH064024B2 - Clostridium butyricum spore production method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing spores in a large-scale culture of Clostridium butyricum.

b. Conventional technology Clostridium butyricum (abbreviated as Cl.hut.)
The group is spore-forming bacteria. Therefore, in order to efficiently perform spore formation of this bacterial group, it is necessary to allow vegetative cells to grow sufficiently. However, even if the vegetative cells are grown sufficiently, it does not always efficiently transfer to spore formation.

In the conventional method, a large amount of calcium carbonate was added to the medium to suppress the sharply decreasing pH,
Spore formation is inefficient because it passes through the pH range in a short time.

c. Problems to be Solved by the Invention The present inventors have conducted diligent research in order to enhance the efficiency of spore formation, and as a result, in order to form spores, there is a certain nutritional source and pH. We have found that it is necessary to keep within a certain range.

Spore generation efficiency can be increased by culturing for a certain specific time within the pH range defined by the present invention.

d. Means for Solving Problems In the present invention, starch is used as a carbon source, and taste liquid, meat extract, peptone, etc. are used as a nitrogen source to prepare a medium, and C
l. but. The present invention provides a method for producing spores of Clostridium butyricum, which comprises inoculating and incubating it with an alkali to maintain the medium at a pH of 6 to 5.5 during the spore formation stage.

The method of the present invention, by artificially controlling the pH with an alkali, maintains a pH range suitable for the growth of vegetative cells, increases the number of vegetative cells, and gradually lowers the pH to the spore formation area, By maintaining this pH range for a long time in the spore-forming area, efficient spore production is achieved.

The present invention can efficiently produce spores by grasping the ecological characteristics of Cl. But. And controlling the pH of the culture.

The spore production method of the present invention is performed, for example, by the process shown in FIG.

For example, at least one starch (including hydrolyzate) such as corn starch, candy starch, soluble starch, and water candy as a carbon source for 30 culture tanks, and a taste liquid (hydrolyzate of soybean) as a nitrogen source. , At least one of meat extract, peptone, etc. is added, sterilized at 121 ° C. for 20 minutes, and cooled to 80 to 90 ° C.

When a culture solution containing Cl. But. Spores that have been previously cultured is added to this medium, the pH of the medium is lowered to about 5.5. Then 37
After cooling to ℃, adjust to pH 7.0. At this time, no calcium carbonate was used to adjust the pH, and 10% NaOH was used.
Or use KOH.

When left at pH 7.0 for 2 to 3 hours, the spores start to grow as nutrient vegetative cells and the pH gradually decreases. This state 3 ~
Hold for 5 hours, during which the pH is adjusted to 7 with 10% NaOH.

Then, the pH is gradually lowered, and when the growth is maximized, the pH is kept at 6.0 for 1 hour to allow the growth to proceed sufficiently, and then the pH is adjusted to 6.0 to 5.5 to adjust to 3 to 6 Hold for time. Thus, vegetative cells can be efficiently obtained.

e. Examples and Comparative Examples As a basal medium, 4% each of cornstarch and taste liquid were used.
The number of spores formed for three types of Cl.but. Was measured when CaCO ₃ was added to the basal medium when the culture was performed with 10% NaOH while controlling the pH.

In the following test, the operation fluid volume was 20, and the spores were measured under a microscope using a Toma hemocytometer.

Example 1. Using the above basal medium, using the strain Cl.but.588,
The pH was lowered while being kept at pH 7, pH 6.5, pH 6.0, 5.7, and pH 5.5 for a certain period of time. Fig. 2 shows the pH control status and pH holding time at this time. The number of spores is 8.2 x 10 ⁸ ce
Complete spores at lls / ml.

Example 2. Using the above basal medium, using Cl.but.703 strain,
The pH was set to pH 7, pH 6.5, pH 6.0, pH 5.7, and pH 5.5, and the pH was lowered while maintaining each pH for a certain period of time. The condition of pH control at this time was exactly the same as in FIG. The number of spores is 7.
It was a complete spore at 0 × 10 ⁸ cells / ml.

Example 3 The pH was controlled in exactly the same manner as in Example 2 using the above basal medium and Cl.but.ATCC19395 strain. The pH control status and pH holding time are as shown in Fig. 2,
The number of spores was 6.8 × 10 ⁸ cells / ml and the spores were complete.

Comparative Example 1. Using the above basal medium and Cl.but.588 strain, the culture solution was first kept constant at pH 7, and then rapidly lowered to pH 5.5. In this case, the pH control for the culture time is the fourth
Described in the figure. The number of spores at the end of culture is 5.0 × 10 ⁷ cells
/ Ml was a complete spore.

Comparative Example 2. Using the above basal medium and the same Cl.but.588 strain, the pH of the culture solution was lowered while being kept at each of pH 7, pH 6.5, and pH 6.0 for a certain period of time. Status of pH control at this time, pH
The holding time is shown in FIG. Spore count is 1.0 × 10 ⁸ cells / ml
Met.

Comparative Example 3. Using Cl.but.588 strain, 1.5% of CaCO ₃ was added to the above basal medium, and pH was not controlled at all. PH at this time
The change with time is as shown in FIG. 6, and the number of spores at the end of culture was 3.0 × 10 ⁸ cells / ml.

Comparative Example 4. Cl.but.588 strain was used, and the pH was not corrected at all with the basal medium alone. The change in pH with time is as shown in FIG. 7, and the number of spores at the end of the culture was 1.2 × 10 ⁷ cells / ml, but it did not progress further at the initial stage of so-called endospores.

Comparative Example 5. Using Cl.but.588 strain, using the above basal medium,
Keeping the pH constant at pH 7 for a long time to grow vegetative cells,
No pH correction was made thereafter. The condition of pH control at this time is as shown in FIG. 8, and the number of spores at the end of the culture was 8.8 × 10 ⁷ cells / ml, which was an endogenous spore and did not transfer to a complete spore.

From the results of these Examples and Comparative Examples, even if the vegetative cells are grown at pH 7, if the pH is not adjusted and the pH is rapidly lowered, the vegetative cells lyse and remain as endospores ( (Fig. 8). Similarly, as shown in Fig. 4, change from pH7 to pH5.5.
Even if H is sharply lowered, complete spores cannot be obtained. The fifth
As shown in the figure, even if the pH is adjusted to 6.0, the spore formation is not so good. However, as shown in Figure 2, pH6.
The number of complete spores is increased by culturing for a long time in the range of 0 to pH5.5. Fig. 6 In the experiment in which CaCO ₃ was added,
Although the number of spores is rather good, it keeps the range of pH 6.0 to pH 5.5 for a long time.

Therefore, as shown in Fig. 2, the spore formation of Cl.but.
It is necessary to control vegetative cells so that vegetative cells proliferate in the region of pH ~ 6.5, and to gradually form spores by gradually decreasing the region of pH 6.0 to pH 5.5. By adding a liquid alkali in this way, it was possible to artificially control the pH, increase the number of spores, and separate spores with a high concentration per unit volume.

f. Effect of the Invention According to the present invention, calcium carbonate in the medium can be removed, and the subsequent steps are simplified. Further, since calcium carbonate, which occupies a large amount as the solid content after culturing, is removed, it is possible to obtain Cl.but.

[Brief description of drawings]

FIG. 1 is a process diagram of the spore production method of the present invention, FIG.
FIG. 3 is a chart showing the pH control state with respect to the culture time in Examples 1 to 3, and FIGS. 4 to 8 are Comparative Examples 1
It is a figure which shows the pH control state with respect to the culture time in ~ 5.

Claims (2)

[Claims] 1. A medium is prepared by using starches as a carbon source and taste liquid, meat extract, peptone, yeast extract, etc. as a nitrogen source, inoculated with Clostridium butyricum and adding an alkali to the culture. A method of producing spores of Clostridium butyricum, which comprises maintaining the medium at pH 6 to 5.5 during the spore formation stage. 2. The method for producing spores of Clostridium butyricum according to claim 1, wherein the medium is maintained at pH 6.0 to 5.5 for 3 to 40 hours.